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Full text of "Pharmacological and toxicological study of heptachlor"

A PHARMACOLOGICAL AND 
TOXICOLOGICAL STUDY OF HEPTACHLOR 



By 
FRANK E. GREENE 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF 

THE UNIVERSITY OF FLORIDA 

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 

DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
February, 1962 



ACKNOWLEDGMENTS 

The author would like to express his sincere appreciation to 
the members of his supervisory committee, Dr. Sidney Cassin, Dr. 
Lauretta Fox, Dr. Melvin Fried, Dr. Thomas Malewitz, and Dr. Elbert 
Voss, Chairman. Their encouragement, advice, and above all, patience 
are gratefully acknowledged. 

The author is indebted to the Graduate Council and the American 
Foundation for Pharmaceutical Education for financial support which 
made possible the completion of this study. 

To his fellow graduate students the author extends special 
thanks for their helpful suggestions, encouragement, and technical 
assistance. 



ii 




I 



• 
• 

• 


TABLE OF CONTENTS 
ACKNOWLEDGMENTS . ...... . . . . 


Page 
ii 
iv 

V 

1 
3 
5 

35 
59 
80 
82 
88 
91 


LIST OF TABLES . . 


CHAPTER 

III. TOXICITY STUDIES 


IV. INVESTIGATION OF THE NATURE OF THL DIFFERENCE IN 

SUSCEPTIBILITY TO THE ACUTE TOXIC EFFECTS OF HEPTACHLOR 
IN MALE AND FEMALE RATS ................ 

V. LOCALIZATION OF THE SITE OF ACTION OF HEPTACHLOR . . . 

VI. SUMMARY AND CONCLUSIONS 


APPENDICES ....... . . . . 


BIBLIOGRAPHY . 

iii 




LIST OF FIGURES 
Figure Pa 8 e 

1. EFFECTS ON THE GROWTH RATE OF VARIOUS CONCENTRATIONS OF 
HEPTACHLOR IN THE DIETS OF MALE AND FEMALE RATS ...... 13 

2. CONTROL LIVER, H AND E, 7 OX 17 

3. LIVER OF FEMALE RAT FED 0.01 PER CENT HEPTACHLOR, H AND E, 

90X • • 19 

4. HIGH MAGNIFICATION OF CONTROL LIVER, H AND E, 350X .... 21 

5. HIGH MAGNIFICATION OF LIVER FROM A FEMALE RAT FED 0.01 PER 
CENT HEPTACHLOR, H AND E, 500X 23 

6. LUNG OF CONTROL ANIMAL SHOWING CHRONIC INFLAMMATORY CHANGES, 

H AND E, 100X 25 

7. THE DOSE-PER CENT MORTALITY CURVE FOR HEPTACHLOR IN MALE 

RATS 30 

8. THE DOSE-PER CENT MORTALITY CURVE FOR HEPTACHLOR IN FEMALE 
RATS 32 



9. THE EFFECT OF SKF-525-A ON THE ACUTE TOXICITY OF HEPTACHLOR 

IN MALE RATS 51 

10. THE EFFECT OF THE CONTROL EMULSION ON THE BLOOD PRESSURE 

AND RESPIRATION OF A PENTOBARBITALIZED DOG 63 

11. THE EFFECT OF HEPTACHLOR ON THE BLOOD PRESSURE AND 
RESPIRATION OF A PENTOBARBITALIZED DOG 64 

12. THE EFFECT OF HEPTACHLOR AND ACETYLCHOLINE ON ISOLATED 
RABBIT ILEUM . 67 




CHAPTER I 



INTRODUCTION 



There is little information in the literature concerning the 
toxicology and pharmacology of Heptachlor (1,4,5 ,6,7 ,8,8-Heptachloro-3a, 
4,7,7a-tetrahydro-4,7 ,-methanoindene) . Frequently the information 
required by law before an insecticide can be marketed is filed with the 
United States Department of Agriculture and with the Food and Drug 
Administration and never published (1). Other observations concerning 
toxicity which may be made during field trials may wind up as progress 
reports of some governmental agency where they are unavailable to a 
literature searcher. Pharmacological studies may be undertaken later 
when hazards of poisoning to warm-blooded animals become apparent, 
pointing to the need for more basic knowledge of the mechanism of action 
of the compound. With this information a rational approach to treatment 
of accidental poisoning and to the development of safe means of 
application of the compound by field personnel may be formulated. 

In 1957, Heptachlor was selected as one of two pesticides to be 
used in the fire ant extermination program undertaken in the Southeastern 
United States. Shortly after initiation of this program it became 
apparent that, in addition to its expected activity against insects, 
the compound was highly toxic to wild and domestic animals. 

Lack of knowledge of the pharmacologic effects produced by this 
compound has forced those treating cases of accidental poisoning to 
resort to empirical methods, which have not always been successful. 



This investigation was undertaken in order to extend the infor- 
mation concerning the pharmacologic and toxicologic properties of 
Heptachlor. It was hoped that this additional information would lead 
to a better understanding of the mechanisms of action of this compound. 
To this end the activity of Heptachlor on the central and autonomic 
nervous system and some factors influencing acute toxicity were studied, 



CHAPTER II 



THE CHEMISTRY OF HEPTACHLOR 



Heptachlor, a member of the cyclodiene family of insecticides, 
was first discovered as a constituent of technical Chlordan, under a 
patent assigned to Hyman (2, p 60). Chemically, it is 1,4,5,6,7,8,8- 
Heptachloro-3a,4,7 ,7a-tetrahydro-4,7 ,-methanoindene. The structural 
formula was shown to be: 




Other members of this family include Chlordan, Aldrin, and 
Dieldrin whose structural formulas are given below. Endrin, a sterio- 
isomer of Dieldrin; Isodrin, a sterioisomer of Aldrin; and Toxaphene, 
a chlorinated camphene mixture, complete the family of cyclodiene 




H CI 



H CI 



Chlordan 




Heptachlor may be prepared by the action of sulfuryl chloride 
on the condensation product of cyclopentadiene and hexachloro-cyclo- 
pentadiene in carbon tetrachloride in the presence of benzoyl peroxide 
(2, p 60). 

All of the cyclodiene insecticides may be prepared by the 
Diels-Alder reaction except Toxaphene, which is made by chlorinating 
camphene to a chlorine content of 67 to 69 per cent (3, p 239). 

Pure Heptachlor is a white crystalline solid, which has a 
melting point of 95-96°C. The technical material available commercially 
contains about 72 per cent Heptachlor and 28 per cent related materials 
and is a soft, waxy solid, light tan in color, with a melting range of 
46 to 74.9 C. Heptachlor is insoluble in water, but soluble in most 
organic solvents (2, p 236). A recrystallized product is also available 
for research purposes, which has a purity of about 90 per cent. 



CHAPTER III 



TOXICITY STUDIES 



Review of the Literature 



The available literature on the cyclodiene group of pesticides 
which does not pertain to agricultural applications is concerned chiefly 
with descriptions of toxic reactions occurring as a result of accidental 
or experimental poisoning in animals. However, a few cases of fatal 
poisoning in humans by Chlordan (4), Aldrin (5, p 5), and Toxaphene (6), 
have been reported. The effects of these compounds on humans appear to 
be identical with those observed in lower animals. No human fatalities 
resulting from Heptachlor poisoning have been reported, but it must be 
considered capable of producing severe toxic effects in humans if 
precautionary measures are not used, as judged by reports of toxicity 
to animals. 

Gross symptoms of acute intoxication following a single exposure 
are essentially the same for all members of the cyclodiene group of 
pesticides. Among these symptoms are signs of central nervous system 
stimulation which may progress to a series of clonic and tonic convulsions. 
Evidence of increased activity of the parasympathetic nervous system is 
usually present. Symptoms reported following acute Heptachlor poisoning 
were increased salivation and lacrimation, generalized tremors, increased 
respiratory rate and volume, violent clonic and tonic convulsions and 
opisthotonus. In terminal stages, respiration was irregular with dyspnea 
and cyanosis as prominent features (7). 






By varying the size and number of doses, three separate types of 
response to Dieldrin have been obtained (5, p 222). A few Large doses 
resulted in one or more convulsions. Unless the animal died, there was 
relatively prompt recovery without permanent damage or great weight loss. 
Many doses of moderate size have produced a complete loss of appetite, 
weight loss, and convulsions. Without treatment, death was seemingly 
inevitable. Many small doses produced one or more convulsions without 
any other apparent effect. 

In addition to these effects, Aldrin (8) and Dieldrin (9) have 
been reported to produce bradycardia, vasodepression, and miosis. 
Lethargy and anorexia are common findings following Aldrin (10), Chlordan 
(5, p 164), Dieldrin (5, p 226) and Endrin (11) poisoning. Partial to 
total blindness, increased response to light tactle stimuli, and 
convulsions induced by auditory stimuli have been attributed to Chlordan 
poisoning (12). These effects have not been noted in cases of poisoning 
by other members of this group. 

It has been shown that absorption of these compounds through the 
skin and mucous membranes in amounts sufficient to produce the toxic 
symptoms previously described, is possible if the proper combination of 
concentration and exposure time is provided (13). 

Unpublished chronic feeding studies (14) have shown that rats 
require about 10 parts per million (ppm) of Heptachlor in their diets 
for production of tissue damage, when maintained at that level for 120 
weeks. Such animals were reported to have manifested a slightly 
depressed rate of growth, but their mortality was no greater than that 
of the controls. However, females maintained on 30 ppm of Heptachlor 



prior to mating had an increased rate of mortality of their offspring. 
Animals given a diet containing 300 ppm died within eleven days, and 
those given 100 ppm showed greater mortality than controls. 

Pathological changes reported in the above study were: degenera- 
tive changes in the cells of the central zone of the liver lobule, kidney 
lesions involving degeneration of the epithelium of the proximal and 
distal convoluted tubules, and some nonspecific changes in the neuronal 
cells of the central nervous system. These effects are essentially the 
same as those reported for other chlorinated hydrocarbons. 

Toxicity data are greatly influenced by the specific experimental 
conditions under which they were obtained. For this reason the results 
of a particular experiment may be meaningless unless the exact experimental 
conditions are also made known. This situation prevails particularly in 
the literature on pesticides where the original data may lie in a file 
of some governmental agency, with only the results, stripped of details 
of experimental conditions, appearing in print. With this in mind, it 
was considered advisable to determine the LDc n of Heptachlor under known 
conditions. 

Since there was such a pronounced sex difference in acute toxicity 
of Heptachlor, it seemed likely that in chronic feeding experiments some 
sex differences might also appear. This aspect seemed worthy of 
investigation and rats of both sexes were maintained on diets containing 
various amounts of Heptachlor for six months. This period of time was 
suggested by Barnes and Denz (15), who felt that any pathology which will 
develop with chronic exposure to a drug should appear within this period. 



Experimental 
Chronic Toxicity Studies 
Materials and Methods 

For chronic toxicity studies, immature rats between 50 and 100 Gms 
were selected. Groups of ten rats of each sex were maintained on ground 
Purina laboratory chow containing 1.0, 0.1, 0.01, and 0.001 per cent 
Heptachlor. One group given ground lab chow alone served as controls. 
The period of feeding was six months for the survivors. During this 
period, the animals were given food and water ad libitum . 

These rats were individually weighed and numbered at the onset 
of the experiment. They were then weighed weekly for the first five 
weeks, at the end of the seventh week, and again at the termination of 
the experiment. At this time they were killed by stunning and exsanguin- 
ation. In addition to the final body weights, organ weights were taken 
for liver, kidneys, and gonads; and these data were analyzed statistically, 
These organs, along with the lungs, adrenals, stomach, esophagus, 
duodenum, colon, ileum, spleen, and heart, were taken for histological 
examination. Rats which died in the course of the experiments were not 
examined histologically as they were frequently mutilated by their cage 
mates or were not found until a number of hours after death. 

After fixation in 10 per cent formalin, all tissues, except those 
used for frozen sections were dehydrated and infiltrated according to 



a 

Rats used in all experiments were NLR (Wistar origin) strain, supplied 

by the National Animal Company, Creve Couer , Missouri. 

b 
Heptachlor used in these and all subsequent studies was supplied by the 

Velsicol Chemical Corporation, Chicago, Illinois, and was labeled 

"recrystallized" Heptachlor, 1009-17. 



the butyl alcohol-paraf in mush method of Johnston et_ aJL. (16). The 
parafin embedded tissues were sectioned at 8 microns and stained with 
Harris hematoxylin and eosin as modified by Malewitz and Smith (17). 

Liver sections were also stained for fat by the oil-red method 
(18, p 124) and for glycogen by the periodic acid-Schiff (PAS) reaction 
(18, p 132). 

Results 

All animals fed Heptachlor in concentrations of 1 or 0.1 per cent 
were dead within two weeks. Body weights, taken at the end of the first 
week of the experiment showed marked reduction in the growth rate of 
these groups, particularly those on the 1 per cent diet. Symptoms of 
toxicity began to appear for these two groups about the sixth day and 
consisted chiefly of lethargy and anorexia with an occasional animal 
showing signs of central nervous system stimulation manifested by 
hyperexcitability and fighting among cage mates. About the eighth day 
this stimulation became more apparent and convulsions of short duration 
were seen frequently. Many of the animals developed lesions around the 
muzzle as a result of fighting with their cage mates. The average 
survival time of these groups is found in Table 1. 

Average body weights taken for all groups are found in Table 2. 
Examination of this table shows a slight depression of growth during 
the first few weeks in the female groups fed the lower concentrations 
of Heptachlor. Male rats fed 0.001 per cent Heptachlor also show a 
slightly depressed rate of growth during the first two weeks. Growth 
curves for the experimental groups surviving the entire period are 



10 



TABLE 1 

AVERAGE SURVIVAL TIMES OF RATS' 

FED HIGH CONCENTRATIONS 

OF HEPTACHLOR 



Diet 


Sex 


Average Survival 
Time (Days) 


17, Heptachlor 


Male 


10.6 


17. Heptachlor 


Female 


9.7 


0.17„ Heptachlor 


Male 


11.9 


0.17. Heptachlor 


Female 


12.6 



All groups contained ten animals, 



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found in Figure 1. When comparing groups of the same sex, this graph 
shows essentially parallel growth for the control and experimental 
groups with the exception of the females maintained on the 0.01 per cent 
Heptachlor diet. When t tests (19, p 164) were performed for the initial 
and final body weights of all groups, this increase in weight was found 
to be significant. Data obtained from statistical analysis of these 
weights are found in Table 3. As a test for significance of all 
comparisons made the confidence limits of 95 per cent probability were 
arbitratily selected. 

Three animals from the group of females fed the 0.01 per cent 
diet died during the course of the experiment, one death occurring during 
the fourth, and two during the sixth months of this study. All animals 
from the other groups survived the entire experimental period. 

On autopsy, no gross pathology was found in any of the organs 
except the lungs, where occasional white nodules were seen. These 
nodules were also found in the control animals and were considered to 
result from pulmonary infection rather than exposure to Heptachlor. 
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weights are found in Table 4. 

These data show that the livers of both male and female rats 
from the 0.01 and 0.001 per cent groups differ significantly from the 
controls. This difference in weight was an increase in all instances 
except for the female group fed the 0.001 per cent diet, where this 
change was a decrease. The kidneys of the 0.01 per cent male group also 
show a significant increase in weight when compared with those of the 
controls. The only significant change observed in gonad weights was an 
increase found in the male group given 0.01 per cent Heptachlor. 



13 




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16 



The most striking changes observed in all tissues studied were 
found in the livers of female rats fed 0.01 per cent Heptachlor. The 
cells of the peri-central vein region were greatly enlarged and pale 
staining. Some cells had undergone karyolysis, while several multi- 
nucleate cells were also seen. Margination of the cytoplasmic material 
and enlarged nuclei were seen in many of the cells in this area. Photo- 
micrographs of control and experimental livers showing these changes are 
found in Figures 2, 3, 4 and 5. Cells of the peri-central vein region 
contained neither fat, nor glycogen. Midzonal cells of sections showing 
the cellular changes previously described showed dense glycogen 
accumulation. In these same sections, fat droplets were prominent in 
the peri-portal regions. These later areas were relatively glycogen poor. 

These changes were present to a lesser degree in female rats fed 
0.001 per cent Heptachlor and were seen only in the 0.01 per cent male 
rat group. The observation of more extensive pathology in female than 
in male rat livers offers a possible explanation for the greater number 
of deaths which occurred in the female group fed 0.01 per cent Heptachlor. 

The lungs of practically all animals, experimental and controls, 
showed evidence of chronic infection. Congestion, lymphocytic infiltra- 
tion, and areas of necrosis were present in varying degrees in all 
animals. Hemosiderin engorged cells, indicative of chronic congestion 
were prominent in many lungs (20, p 56). A photomicrograph showing some 
of these changes is found in Figure 6. 






The author would like lo express his appreciation to Drs. J. E. Edwards 
and M. Waid of the Department of Pathology, J. Hillis Miller Health 
Center, for their valuable suggestions and interpretations of the 
pathology found in the livers of these animals. 






FIGURE 3. LIVER OF FEMALE RAT FED 0.01 PER CENT 
HEPTACHLOR, H AND E, 9 OX 



20 



» 







FIGURE 5. HIGH MAGNIFICATION OF LIVER FROM A FEMALE RAT 

FED 0.01 PER CENT HEPTACHLOR 
H AND E, 500X 



< 



24 







• 


■ 


v 


• 
• 


' 


-* ■ ^^k ^^^^^A • mm* ' 



**** 



'*:>,. I 



J 



• 



v. 
P 



*n 



*•* 










FIGURE 6. LUNG OF CONTROL ANIMAL SHOWING CHRONIC 

INFLAMMATORY CHANGES, M 

H AND E, 100X ^ 



i 



26 







27 



The results of histological examination of the other tissues and 
organs were non-remarkable. 

Acute Toxicity Studies 
Determination of the Oral LD50 of the Corn Oil Solution of Heptachlor 

Materials and Methods 

Preliminary investigation had shown the dosage range for to 
100 per cent fatalities in male rats to be from 40 to 100 mg/Kg, and in 
females from 60 to 190 mg/Kg. Doses at graded increments were administered 
orally to groups of eight rats each using a Phipps and Byrd oral needle 
following a period of fasting of 12 to 18 hours. The rats had free 
access to water during the period of fasting and were given food 
immediately following the administration of Heptachlor. These rats were 
then observed for a period of eight days, and the LD50 was calculated 
from the number of deaths occurring during this period using the method 
of Litchfield and Wilcoxon (21). 

The general procedure as outlined by this method is followed 
in the calculation of the LD50 f° r tne male rats found in Appendix III. 
For determination of the female LDcq, only the results are shown. 

Results 

Deaths resulting from a single oral dose of Heptachlor usually 
fell into two categories: 1) those dying within the first 24 hours, 
and 2) those dying from about the fifth to the eighth day. These 
intervals represent the period of early and delayed toxicity respectively. 



The method used for the preparation of the corn oil solution is found 
in Appendix I. 



28 



In both periods the symptoms prior to death were similar and consisted 
of a series of short spasms which appeared in increasing frequency, 
ultimately terminating in a clonic and tonic convulsion. Following these 
convulsions, the muzzles of all animals were wet, indicating that an 
increase in secretion had occurred during the seizure. This finding was 
indicative of parasympathetic nervous system stimulation, and was seen 
following convulsions only. The LJ^o's and their confidence limits are 
given below. 

Male rats .- The data obtained from the determination of the LD50 
are given in Table 5. The graph for the data is found in Figure 7. The 
LD5O and its 19/20 confidence limits calculated from these data is 59 
(49 to 71) mg/Kg. 

Female rats .- The data obtained from this experiment are found 
in Table 6, and presented graphically in Figure 8. The calculated LD50 
and its 19/20 confidence limits is 132 (114 to 154) mg/Kg. 

Discussion 

The LD50 values determined for male rats are in reasonable 
agreement with those previously reported (14). The periods of early and 
delayed toxicity as described by Lehman (13) were also seen in this study, 
As in previous studies (14) male rats proved to be much more susceptible 
to a single oral dose of Heptachlor than females. The nature of this 
difference is investigated in the following chapter. 

The difference in susceptibility of male and female rats was 
reversed in the acute and chronic toxicity studies. While males were 
more susceptible to the acute effects of Heptachlor, females were more 



29 



» 



TABLE 5 

SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL 
SOLUTION OF HEPTACHLOR IN MALE RATS 



> 







Observed* 


Expected 


Observed 


Contributions 


Dose 


Killed 


Per Cent 


Per Cent 


Minus 


t0 2 
(Chi) Z 


mg/Kg 


Tested 


Mortality 


Mortality 


Expected 


40 


0/8 


4.4 


13 


8.6 


0.060 


50 


1/8 


12.5 


34 


21.5 


0.190 


60 


4/8 


50.0 


52 


2.0 


0.002 


70 


5/8 


62.5 


68 


5.5 


0.025 


80 


7/8 


87.5 


81 


6.5 


0.028 


100 


8/8 


98.4 


93.5 


4.9 


0.039 



> 



The observed values listed for and 100 per cent effect 
represent corrected values obtained by the Litchfield and Wilcoxon 
method. 

The expected values were obtained from Figure 7. 





30 




99.9 


" 1 ' | ' | ' ' | ' ' ' ' 






99 


o / 




> 










95 


- / - 






90 


/ — 






>- 


/ 






H 


(y 






_i 


- I — 






< 


I 






t- 


1 






<r 


I 






o 


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2 


r 






h- 


1 






5 50 


— / 




1 


o 

cr 

UJ 


1 






a. 


/ ° 






10 


~ — 




> 


I 


/ ° 

• 1 i 1 i 1 i i 1 i i i i 




20 30 50 70 100 200 




DOSE, MG/KG 




Fig. 7. --The dose-per cent mortality 




curve for Heptachlor In male rats. 



31 



TABLE 6 

SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL 
SOLUTION OF HEPTACHLOR IN FEMALE RATS 







Observed 


b 
Expected 


Observed 


Contributions 


Dose 


Killed 


Per Cent 


Per Cent 


Minus 


t0 2 
(Chi) Z 


mg/Kg 


Tested 


Mortality 


Mortality 


Expected 


60 


0/8 


0.6 


1.8 


1.2 


0.008 


80 


1/8 


12.5 


9.0 


3.0 


0.011 


100 


1/8 


12.5 


22.0 


9.5 


0.050 


120 


2/8 


25.0 


39.0 


14.0 


0.080 


140 


4/8 


50.0 


55.0 


5.0 


0.010 


150 


7/8 


87.5 


62.0 


25.5 


0.275 


160 


6/8 


75.0 


70.0 


5.0 


0.011 


170 


8/8 


92.6 


74.0 


18.6 


0.180 



The observed values listed for and 100 per cent effect 
represent corrected values obtained by the Litchfield and Wilcoxon 
method. 



'The expected values were obtained from Figure 8. 





32 




95 


ii ii i i i i I 






90 


^_ / ___ 




1 




/ 

o/ 






50 


— >0 — 






>- 
_) 

cr 
o 

2 


/ ° 




1 


l- 


O/0 




^ 10 


— / 






PERI 


i 1 i . , i 






0.1 


— / — 




> 




' 






0.01 


1 1 — i — 1 i i 1 i i i i 1 






40 70 100 200 300 




DOSE, MG/KG 




Fig. 8. — The dose-per cent mortality 
curve for Heptachlor in female rats. 



33 



susceptible to chronic exposure to Heptachlor. The only pathologic 
differences observed in the surviving animals was a more extensive liver 
damage in female rats, when compared to males fed the same concentration 
of Heptachlor. If the death of the three animals in the female group 
fed 0.01 per cent Heptachlor resulted from this type of damage, it is 
possible that the lower mortality rate observed in male rats fed the 
same diet could be due to some protective action of this organ by 
testosterone. This protective role of testosterone has been proposed 
by Seyle (22). The significant increase in the weight of the testes of 
rats fed 0.01 per cent Heptachlor could then represent a functional 
response to an increased utilization of testosterone. This event could 
arise following an increased demand of long duration for this hormone 
if it should be involved in such a role in the liver. 

The over-all activity of Heptachlor falls into two, apparently 
unrelated, categories: 1) central nervous system stimulation seen in 
acute poisoning with a single large dose, or frequently administered 
smaller doses, and 2) pathological changes in organs such as the liver 
and kidney, following continuous exposure to small amounts of this 
compound. The period of delayed toxicity may contain elements of both 
types of action. 

The anorexic effect of Heptachlor and the property of lipids of 
the central nervous system to resist mobilization in the face of 
protracted starvation suggested the following sequence of events in the 
period of delayed toxicity of Heptachlor. 

Following ingestion of Heptachlor in an amount less than the 
quantity necessary to cause death in the early toxicity period, 



34 



Heptachlor and the epoxide are stored in body fat. During the period of 
anorexia which may develop, the animal will mobilize this fat when 
glycogen deposits are depleted, but lipids of the central nervous system 
would not be involved in this process. Heptachlor, which is stored in 
body fat would be mobilized as the fat in which it is stored is broken 
down. The Heptachlor displaced in this manner would tend to relocate 
in other lipid tissue. The unmobilized lipid of the central nervous 
system is a likely site of such events. Through such a process Heptachlor 
concentration in the nervous system could gradually build up, leading to 
the symptoms of central nervous system stimulation and ultimately, 
convulsions in these animals. 

A logical treatment of Heptachlor poisoning should be directed 
toward the two phases of the action of Heptachlor: 1) control of the 
convulsions during the period of early toxicity, and 2) prevention of 
the secondary toxic effects. Control of the later effects could possibly 
be accomplished by supportive therapy directed toward protection of the 
liver and other organs from further damage. A low fat, high protein 
diet, plus one or more lipotropic substances (23, p 928), is recommended 
for treatment of damaged livers, and would, at the same time provide 
nutritional support for the animal. Such a regimen would be expected 
to prevent mobilization of body fat and secondary deposition of Heptachlor 
in the central nervous system, if this event should actually take place. 
The central nervous system effects could probably be controlled by 
cautious administration of barbiturates. 



CHAPTER IV 



INVESTIGATION OF THE NATURE OF THE DIFFERENCE IN 

SUSCEPTIBILITY TO THE ACUTE TOXIC EFFECTS OF 

HEPTACHLOR IN MALE: AND FEMALE RATS 



Review of the Literature 
In the course of work directed toward determination of tissue 
residues from Heptachlor, Davidow and Radomski (24) found a metabolically 
altered derivative, an epoxide, along with Heptachlor in the fat of 
Heptachlor-f ed dogs. They reported the following as the structure of 
this compound: 




H CI 



These authors stated that this was a type of biological oxidation product 
which has not previously been reported, and suggested that it may be a 
stable intermediate of the process of biological hydroxy lation which, 
due to its solubility in fat, tended to accumulate in adipose tissue. 
Since this initial discovery, the epoxide of Heptachlor, along with the 
parent compound, has been isolated and identified in the body fat of 
rats and rabbits (25), (26), and in the whole insect in the case of the 
housefly, Musca domesticus (27). The female rat stores the epoxide to 
a much greater extent in fat than does the male, the biological 



35 



36 



a 
multiplication ratio being 6.2 for the female and 1.2 for the male 

when both are maintained on 30 ppm of Heptachlor in their diet (25). 

A particularly interesting aspect of the toxicity of Heptachlor 
in the rat is the difference in response of the male and female to a 
single oral dose, the reported LDcq for females being 142 mg/Kg, and 
that for males, 60 mg/Kg (14). A possible explanation for this differ- 
ence could be a different rate of conversion of Heptachlor to its 
epoxide, a compound which has an intravenous toxicity distinctly greater 
than the parent compound. When given a dose of 10 mg/Kg, 100 per cent 
of epoxide treated mice died, while Heptachlor at the same dosage level 
caused no deaths (25). If male rats were capable of converting Heptachlor 
to its epoxide at a faster rate, this might explain the higher mortality 
produced in males than females given the same dose. 

Similar differences in sex responses have been observed with 
other compounds, particularly the barbiturates, in which it was noted 
that male rats consistently slept for shorter times than females given 
the same dose of pentobarbital. Jarcho (28) noted that this sex 
difference could be demonstrated only with the barbiturates which are 
known to be detoxified in the liver. Hoick et a_l. (29) investigated 
the effects of testosterone propionate and estradiol dipropionate 
administered prior to hexobarbital treatment in normal male and female 
rats and was able to shorten the sleeping time of testosterone-treated 
females to that of the males. They also were able to reduce the sleeping 



The biological multiplication ratio is a measure of the ability to 
concentrate a substance in the body fat and is calculated by dividing 
the concentration of the substance in the diet into the concentration 
of the substance found in the fat. 



37 



time in the testosterone-treated male below that of the nontreated 
control males. Cameron e_t_ a_l. (30) reported an increase in mean 
barbiturate-induced sleeping time following castration in male rats. 
Tureman et al_. (31) found that gonadectomy increased sleeping time for 
both sexes, and that testosterone given to male gonadectomized rats and 
intact females decreased sleeping time. Changes in sleeping times have 
provided a convenient method for the determination of the effects of 
various experimental procedures on metabolic transformations which 
resulted in the inactivation of certain barbiturates. Conditions which 
produce changes in sleeping time may also be expected to affect the 
rate of biotransformation of other substances whose metabolism does not 
provide so convenient an endpoint . 

Quinn et_ a_l. (32) investigating the biotransformation of 
hexabarbital by the microsomal fraction from rat liver homogenate, found 
close correlation between the rates of biotransformation and the sleeping 
time. Female rats slept four to five times as long as males and showed 
a correspondingly lower rate of biotransformation. Further, this 
difference in the sexes was reduced by administration of testosterone 
to females for six weeks prior to testing, producing both an increase 
in biotransformation and a corresponding shortening of sleeping time. 

In addition to these drugs, sex differences in the rates of 
metabolism in rats have been noted with two non-chlorinated hydrocarbon 
insecticides, Schradan and Parathion (33). These compounds are 
relatively weak cholinesterase inhibitors in vitro , but are converted 
in vivo to powerful cholinesterase inhibitors, Schradan was found to 
be more toxic for males and Parathion for females, The liver of the 



38 



male rat has been shown to convert Schradan to its more active oxide 
better than the livers of females; the reverse holds true for Parathion, 
which is converted to paraoxone by the liver of the females more rapidly 
than in that of the male. These reactions were found to occur in the 
microsomal fraction of liver homogenate and to be DPN and Mg dependent. 
Male and female rats are equally susceptible to the cholinesterase 
inhibitor TEPP, which does not undergo conversion in_ vivo . 

Hoick et_ al_. (34) produced hypothyroidism in rats of both sexes 
by using 200 mg of propylthiouracil mixed with 1 Kg of food. The animals 
were maintained on this diet for 20 days prior to testing. This 
experimental hypothyroidism was expected to decrease liver metabolism 
and thereby prolong the action of those barbiturates metabolized in 
this organ. Using pentobarbital, he was able to produce an increase in 
sleeping time in both sexes, but a significant difference between the 
male and female response was still present. 

The Smith, Kline and French Laboratories have synthesized a 
compound which was found to be capable of greatly increasing the effects 
of certain drugs by affecting the rate at which they are degraded in the 
body. This compound, beta-diethylaminoethyl-diphenylpropyl acetate, was 
given the generic name of diphenylpropylacetate and referred to as 
SKF-525-A. It was sent to the National Institutes of Health where its 
effects on the biotransformation of a variety of drugs were studied. 
Axelrod e_t_ al. (35) testing the effects of pretreatment with SKF-525-A 
on hexabarbital sleeping time, found it to be increased four times over 
that of the saline-treated controls. He also followed blood levels of 
the barbiturate during this period and found the half-life paralleled 



39 



this increase. Blood levels of hexobarbital were measured in animals 
at the time they regained their righting reflex and were found to be the 
same regardless of whether the animal had been pretreated with SKF-525-A 
or not. In addition, animals recovering from anesthesia cannot be 
reinducted by use of SKF-525-A as is the case with chlorpromazine or 
glucose, which are classified as potentiators (35). On the basis of 
these findings, SKF-525-A was judged to be a "prolonging" agent rather 
than a potentiator. 

Cook et al. (36) determined that the maximum effect of hexobarbi- 
tal sleeping time was obtained when SKF-525-A was given 40 minutes prior 
to administration of the drug. He also found SKF-525-A to be equally 
effective when given either intraper itoneally or orally. He was able 
to produce a 35-fold increase in the sleeping time of rats pretreated 
with SKF-525-A over saline treated controls. In other studies, Cook 
et_ al. (37) found that SKF-525-A had no effect on thiopental, ether, or 
nitrous oxide anesthesia, or on the sleeping times of barbital, or 
methylparafynol. None of these substances are inactivated by metabolism. 
However, SKF-525-A did enhance the analgesic properties of morphine 
sulfate, codeine phosphate, and methorphinan, and prolonged sleeping 
times of Seconal, Amytal, Butethal, Ortal, pentobarbital and chloral 
hydrate. Metabolism of members of the later group results in loss of 
activity. 

In vitro studies by Cooper (38), using liver homogenates 
demonstrated the inhibition of a variety of biotransformation reactions 
with SKF-525-A. Some of these were: side chain oxidations, dealkylations , 
deaminations and cleavage of ether linkages. La Du (39), investigating 



40 



a group of compounds whose biotransformations could be accomplished 
with a system comprised of liver microsomes plus reduced TPN and oxygen, 
found that all could be inhibited by SKF-525-A. The conversion of 
Parathion and Scaradan to their oxides, cited previously (33), and 
found to be DPN dependent was also inhibited by SKF-525-A. 

Cooper (40) tested the effects of SKF-525-A on enzyme systems 
involved in generating reduced TPN and in transporting hydrogen from 
reduced TPN to oxygen via the cytochrome system. He also tested effects 
on DPN-requiring enzymes, using the alcohol dehydrogenase system. He 
was unable to demonstrate inhibition in either case and concluded that 
the action of SKF-525-A in_ vivo probably lay in its effect on some 
other, as yet unidentified, system common to all of the biotransformation 
reactions studied. 

Since the discovery of these properties of SKF-525-A, two other 
compounds have been found which inhibit the same biotransformation 
reactions. They are 2 ,4,-dichloro-6-phenoxyethyl diethylamine (Lilly 
18947) and iproniazid. Studies on the effects of these compounds on 
hexobarbital sleeping time by Fouts and Brodie (41), (42) have shown 
Lilly 18947 to be most effective, followed by SKF-525-A and iproniazid, 
in that order. 

Recently, workers at the Lilly Laboratories have synthesized 
another compound designated as Lilly 32391 (2 phenyl-4,6-dichloro-phenoxy) 
ethylamine HCl, which is said to be about ten times as potent as Lilly 
compound 18947 (43). The structures of these compounds are shown in 
Table 7. 





41 



TABLE 7 

STRUCTURAL FORMULAS OF SOME INHIBITORS 
OF DRUG METABOLISM 



/? / C 2 H 5 

C-0-CH 2 -CH 2 -N 

C \ X °2 H 5 

V CH 2 -CH 2 -CH 3 



0=C-NH-NH-CH-CH, 
CH, 



t S> 



SKF-525-A 



Ipronlazld 



^^ 




•HBr 



92 H 5 



l-f VO-CH 2 -CH 2 -N-C 2 H 5 



^^ 



CI-/ Vo-CH 2 -CH 2 -NH 2 -HCI 



Lilly Compound 18947 



Lilly Compound 32391 



42 



Metabolic activation of Heptachlor rather than detoxification is 
strongly suggested by data from the experiments previously discussed 
concerning the nature of sex difference in response to certain barbitu- 
rates. Since male rats are capable of metabolizing barbiturates at a 
faster rate than females, it is not unreasonable to assume that similar 
differences in the metabolism of other compounds may exist. If Heptachlor 
falls into this category, the difference in response by male and female 
rats could be explained by a more rapid rate of conversion, in the male, 
to a compound more toxic than the original one. The only known metabolite 
of Heptachlor, its epoxide, is in fact more toxic than the parent 
compound. In view of these facts, it was felt that investigations 
analogous to the studies of sex differences in barbiturate response 
could yield valuable information concerning some of the factors involved 
in the mechanism of the toxic action of Heptachlor. 

Experimental 
The Effect of Gonadectomy and Gonadal Hormones on Acute Toxicity 
Materials and Methods 

To determine the effects on the acute toxicity of Heptachlor 
produced by castration and the administration of exogenous hormones to 
castrated animals, thirty male and thirty female rats weighing 150 to 
200 Gms were castrated by the methods of D'Amour and Blood (44, p 44, 45) 
and allowed one month to recover. At this time, the separate groups of 
males and females were each divided into three sub-groups of ten animals; 
and to these, one normal (uncastrateu) group of each sex was added for 
controls. 



43 



Daily hormone or sesame oil injections were given subcutaneously 

according to the schedule given below. These injections were begun two 

weeks before and continued one week following administration of 

Heptachlor. The dosage regimen selected was one used by Hoick et al. (29) 

in their studies on sex differences in pentobarbital sleeping time in 

rats. 

Group Treatment Dosage 

Non-castrate Sesame oil 1 ml/Kg/day 

Castrate Sesame oil 1 ml/Kg/day 

Castrate Estradiol dipropionate 1 mg/Kg/day 

Castrate Testosterone propionate 1 mg/Kg/day 

On the fourteenth day of treatment, the rats were weighed and 

given an oral dose of 200 mg/Kg of Heptachlor. These rats were older 

and heavier than those used in the determination of the LDcq, and 

preliminary trials had shown them to be more resistant to the toxic 

effects of Heptachlor than younger rats. These trials had indicated 

that at a dosage level of 200 mg/Kg, one could expect about 80 per cent 

fatality in males and 40 per cent fatality in females. The animals 

were observed for fourteen days following administration of Heptachlor 

and all deaths were recorded. Dead animals were autopsied, and the 

seminal vesicles of the males, and uteri of the females, were removed 

and examined for gross effects. These organs were not examined 

histologically. 




The testosterone used in these studies was manufactured by Charles F. 
Pfizer and Company, Brooklyn, New York, under the trade name Synandrol 
(lot no. 88354). Estradiol was supplied by Ciba Pharmaceuticals 
Incorporated, Summit, New Jersey, as their trade name product, Ovocylin 
(control no. 242156). 




44 




Results 

The data from this experiment are found in Table 8. The expected 
sex difference appeared in the non-castrate group with twice as many 
males as females dying during the first day. Testosterone increased 
the toxicity of Heptachlor for castrate males and females to the level 
of normal males and shortened the length of time necessary for symptoms 
of toxicity to appear. All estradiol-treated animals escaped the early 
period of toxicity, but some deaths did occur in both male and female 
rats beginning on the fourth day. At the end of the first week, the 
sesame-oil-treated, castrate animals showed the same malerfemale death 
ratio as the non-castrate, sesame-oil-treated animals; although the 
actual number of fatalities was less in the former group. During the 
second week, several fatalities occurred in the castrate sesame-oil- 
treated and in the castrate, estradiol-treated groups. These deaths 
were delayed longer than the expected period of delayed toxicity which 
usually lasts from about the fourth to the eighth day. In this 
particular experiment, however, this period was very mild for the 
control animals with only an occasional animal showing signs of central 
nervous system stimulation. No deaths occurred among the control groups 
during this period. By the end of the second week, the total number of 
deaths was essentially the same for all groups except the non-castrate, 
sesame-oil-treated female group. The latter group had the lowest number 
of fatalities during the experimental period. 

All animals receiving testosterone or estradiol lost weight 
during the two week period prior to the administration of Heptachlor. 
Sesame-oil-injected animals showed a slight increase in body weight 






45 






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46 



during this period. These changes will be tabulated along with the 
weight changes of other experimental groups at the end of the chapter. 

The Effect of Hormone Administration on Acute Toxicity 
of Heptachlor in Normal Rats 

Materials and Methods 

This experiment was undertaken in order to determine if the 
effects observed in castrate animals could be demonstrated in normal 
animals. An additional point of investigation was to determine if 
Nilevar (17-ethyl-19-nortestosterone) had any demonstrable effect on the 
toxicity of Heptachlor. Nilevar is considered to be an anabolic steroid 
as it has an anabolic : androgenic ratio of 20; whereas this ratio for 
testosterone is about one (45, p 893). This point of investigation arose 
when it was observed in the previous experiment that testosterone greatly 
increased the toxicity of Heptachlor in castrate rats. If this activity 
was due to an anabolic rather than androgenic effect, Nilevar could be 
expected to increase toxicity, also, as it shares the anabolic activity 
of testosterone while having only a fraction of its androgenic activity. 
The experimental groups and their dosage regimen is listed below: 

Group Treatment Dosage 

I Sesame oil 1 ml/Kg/day 

II Estradiol dipropionate 1 mg/Kg/day 

III Testosterone priopionate 1 mg/Kg/day 

IV Nilevar 3 1 mg/Kg/day 

The same series was set up for each sex, and each group contained 

8 animals. The injections were given subcutaneously every day according 



a The Nilevar (control no. 2041) used in this experiment was manufactured 
by G. D. Searle and Company, Chicago, Illinois. The other hormone 
preparations were the same as those used in the previous experiment. 



47 



to the schedule previously listed. They were given two weeks before 
and one week after 200 mg/Kg of Heptachlor was given orally. These 
animals were observed for a period of two weeks after the administration 
of Heptachlor, and deaths were recorded as they occurred. Dead animals 
were autopsied, and gonads and accessory sex organs were removed and 
examined grossly for effects produced by their particular treatment. 

Results 

When compared with controls, the data from this experiment, found 
in Table 9, do not show a clear-cut effect resulting from the administra- 
tion of hormones to normal animals. Group I, the sesame-oil-treated 
controls, showed the normal pattern of male and female toxicity; but 
the delayed interval of the acute toxicity period occurred later than 
usual in the female group. 

Estradiol appeared to have increased the early toxicity of 
Heptachlor in male rats. Deaths due to delayed toxicity developed more 
rapidly in females treated with this hormone than in the sesame-oil- 
treated group. 

During the first day mortality was much higher in the testoste- 
rone-treated males than in other groups. Four animals from this group 
died; while only one death occurred in all other groups combined. At 
the end of the experimental period, all animals from this group were 
dead. Toxic symptoms appeared sooner in the testosterone-treated 
females than in the controls. Symptoms of the former group began on 
the third day and lasted until the tenth day. 

The pattern of toxicity of Heptachlor in male rats which were 
pretreated with Nilevar differed from that of other experimental groups. 



48 



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49 



Normally, most of the fatalities which will occur in a group of animals 
given a standard dose of Heptachlor will fall in a two or three day 
period. Nilevar-treated animals, however, did not follow this pattern. 
Deaths occurred in this group from the first through the tenth day 
following Heptachlor. Mortality of females given this hormone before 
Heptachlor was slightly lower than in the female control group,, 

Gross examination of the gonads and accessory sex organs of 
those animals which died during the course of the experiment showed a 
consistent enlargement and fluid distention of the seminal vesicles of 
the testosterone-treated males and atrophy of these structures in animals 
receiving estradiol* Uteri of estradiol-treated animals exhibited 
increased vascularity when compared to controls; while uteri from the 
testosterone-treated groups were uniformly pale. No effects on either 
of these tissues were seen in the Nilevar-treated animals. 

Changes in body weight will be tabulated along with the weight 
changes of other experimental groups at the end of this section. 

The Effect of SKF-525-A on the Acute Toxicity 
of Heptachlor in Male Rats 

Materials and Methods 

This experiment was undertaken in order to test the hypothesis 

that Heptachlor was metabolized to a more toxic product; and if this 

metabolism could be slowed or stopped, toxicity would be decreased. 

SKF-525-A was selected for this experiment because of its known ability 

to inhibit the metabolism of a variety of compounds. 



SKF-525-A was supplied by the Smith, Kline and French Laboratories, 
Philadelphia, Pennsylvania. 



50 



Twenty-four, 150 to 200 Gm male rats were divided into three 

groups of eight rats each and were given the following treatment: 

Groups Treatment 

A Water 10 ml/Kg 

B SKF-525-A 100 mg/Kg 

C SKF-525-A 100 mg/Kg every twelve hours 

This treatment was given orally about forty minutes before the oral 

administration of 150 mg/Kg of Heptachlor, This period of time was 

chosen because SKF-525-A has been shown to give maximum prolongation of 

hexabarbital sleeping time forty minutes after ingestion (36). Since 

this same work had shown SKF-525-A to be active as long as fifteen hours 

after administration, one group was given this compound every twelve 

hours to determine if additional benefits could be gained by multiple 

dosage. All animals were observed for a period of eight days, and 

deaths were recorded. 

Results 

The graph for the data from this experiment is found in Figure 9. 
Prior treatment of SKF-525-A was shown to produce a marked delay of the 
onset of toxic symptoms when compared with water-treated controls. 
Examination of the graph shows that some additional protection was 
obtained by administration of SKF-525-A every twelve hours. Five 
animals from the singly treated group died after the first twenty-four 
hours; while only two deaths occTurred in the group given SKF-525-A 
every twelve hours. 









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52 



The Effect of Propylthiouracil (PTU) on Acute Toxicity 
Materials and Methods 

This experiment was undertaken in an effort to determine if PTU 
induced hypothyroidism, and resulting decrease in metabolic activity (34) 
would effect the acute toxicity of Heptachlor in rats. Twenty-four 
animals of each sex were placed in three groups of eight rats each, 
body weights recorded, and two groups of each sex were given 0.15 per 
cent PTU in ground Purina laboratory chow ad libitum for a period of 
three weeks. As a control, the remaining group of each sex was fed 
ground laboratory chow, alone. At the end of this period, the animals 
were weighed, and one group of the PTU fed and the control fed group 
from each sex was given Heptachlor 200 mg/Kg orally The remaining PTU 
group from each sex was given corn oil, 5 ml/Kg and observed for possible 
toxic effects which might have been produced by PTU. After administration 
of Heptachlor, the animals were observed for a period of eight days, and 
deaths were recorded „ At death, the animals were autopsied and the 
thyroid glands removed and examined grossly for the effects of PTU. 

Results 

Toxic symptoms developed rapidly in both male and female rats 
which had been maintained on PTU prior to the administration of Heptachlor. 
All males from these groups were dead within one and one-half hours. 
Females from the PTU-Heptachlor group developed symptoms of central 
nervous system stimulation within one hour, and seven were dead in less 
than twenty-four hours. 



a The propylthiouracil used for this experiment was obtained by crushing 
50 mg tablets (control no. Y 563J) manufactured by Parke, Davis, and 
Company, Detroit, Michigan. 



53 



Animals fed regular laboratory chow developed symptoms more 
slowly, although all males from this group died within twenty-four hours. 
Only two rats from the female group given regular laboratory chow died 
within the eight-day observation period. No deaths occurred in the 
groups fed the PTU diet and given corn oil. 

The thyroids of PTU-treated animals which died during this 
experiment appeared to be greatly enlarged when compared to non-PTU-fed 
controls. All male rats, including controls, lost weight during the 
experimental period. Both groups of females given PTU in their diet 
showed a weight reduction, while female rats given the control diet 
gained weight. Changes in body weight will be tabulated along with the 
weight changes of other experimental groups at the end of this section. 

Discussion 

Evidence of a possible endocrine basis for the difference in 
sex response is presented in Table 10. Castration decreased the number 
of early deaths in both male and female rats. However, at the end of the 
experimental period, there was little difference between these groups and 
the non-castrate males which characteristically demonstrate the highest 
percentage mortality for a given dose of Heptachlor. Data in this table 
show only slight differences between any experimental groups at the end 
of the two week period of observation. Normal females still had fewer 
deaths than other groups, except for females given Nilevar. The latter 
group had one less fatality than the control females. 

The rate at which the toxic effects of Heptachlor appeared was 
accelerated in both castrate and normal animals treated with testosterone. 



54 



TABLE 10 

COMBINED DATA FROM EXPERIMENTS CONCERNING THE 
ENDOCRINE INFLUENCE ON THE ACUTE 
TOXICITY OF HEPTACHLOR IN RATS 



Number of Deaths on Day Indicated 

Following Oral Administration 

of Heptachlor, 200 mg/Kg 



( 


Jroup 






























Killed 
Tested 


Per Cent 
































Killed 






1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


11 


12 


13 


14 






Female 


I 


a 
Normal 




















2 


2 


1 

















5/8 


62.5 


II 


Normal 











2 





2 


1 


1 


1 

















7/8 


87.5 


III 


Norma 1 








1 


1 


1 





1 





1 


1 














6/8 


75.0 


IV 


Normal 








2 














1 


1 

















4/8 


50.0 


V 


Norma 1 


3 



































1 





4/8 


50.0 


VI 


_ a 
Castrate 














1 


1 











2 


2 








1 


7/8 


87.5 


VII 


Castrate 











1 


2 








1 











1 





2 


7/8 


87.5 


VIII 


Castrate 


7 









































7/8 


87.5 


















Mai 


e 


















I 


a 
Normal 





4 


3 



































7/8 


87.5 


II 


Norma 1 


1 


5 






































6/8 


75.0 


III 


Normal , 
Normal 


4 


3 




















1 

















8/8 


100.0 


IV 


2 








1 


1 


1 











2 














7/8 


87.5 


V 


Normal 


6 









































6/8 


75.0 


VI 


Castrate 
Castrate 


2 











1 


1 





2 














1 





7/8 


87.5 


VII 














1 


2 


1 


1 





1 














6/8 


75.0 


VIII 


Castrate 


6 














1 


























7/8 


87.5 



a Plus Sesame Oil, 1 ml/Kg/day 
b, 



Plus Estradiol Dipropionate, 1 mg/Kg/day 
Plus Testosterone Propionate, 1 mg/Kg/day 
Plus Nilevar, 1 mg/Kg/day 



55 



This effect was prominent in castrate male and female rats, while in 
normal rats this response was not as well defined. These observations, 
together with a slower development of toxicity in the castrate male, 
suggest that an active role is played by the gonadal hormones of male 
rats in the acute toxicity of Heptachlor. An additional observation 
favoring this explanation of the role of male hormones is the significant 
increase in the testicular weight of male rats fed 0.01 per cent 
Heptachlor, presented in Chapter III. Since no pathological changes 
were found in these organs, this increase in weight could have resulted 
from a physiological hypertrophy caused by an increased demand for 
gonadal hormones. Such a phenomenon could result from continuous demand 
for these hormones in the metabolism of Heptachlor. A change in weight 
has not been previously reported following chronic exposure to Heptachlor, 

Estrogenic effects on the toxicity of Heptachlor appear to be 
inconsistent. Castration increased the number of deaths in female rats, 
but estradiol administered to castrate females failed to prevent this 
increase in mortality. 

Changes in body weight observed in the experimental animals from 
both hormone experiments along with changes which were recorded for 
propylthioracil-treated animals are listed in Table 11. It was observed 
that some of the procedures used in these experiments produced a weight 
loss while other procedures increased body weight. A weight change 
brought about by a change of the size of the lipid depot of the body 
could alter the space available for storage of Heptachlor and its 
metabolite. This, in turn, could effect the susceptibility of an animal 
to this compound. However, comparison of the weight changes and the 






56 



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number of mortalities observed in all groups did not show any relation- 
ship between weight changes and toxicity. 

The delaying effects of SKF-525-A on the appearance of toxic 
symptoms provides support for the theory of metabolic activation of 
Heptachlor. It is difficult to assess the actual benefit obtained by 
repeated administration of SKF-525-A, due to a higher initial number of 
deaths in the group which was given this compound every twelve hours. 
As a result of this there were fewer animals to observe in this group 
later in the experiment than in the group which only received a single 
treatment of SKF-525-A. 

The opposing effects of SKF-525-A and testosterone on the 
metabolism of certain drugs, as demonstrated by hexobarbital sleeping 
time studies, were also seen during these experiments. The effect of 
these compounds were reversed in their influence on Heptachlor toxicity, 
as testosterone increases toxicity while SKF-525-A delays or decreases 
toxicity. The opposite holds true for activity of hexobarbital, which 
is inactivated by metabolic pathways. In the later case, testosterone 
shortens the activity, while SKF-525-A prolongs the effects of this 
drug. Since inhibition of drug metabolism has not been reported for 
testosterone, nor stimulation of this process by SKF-525-A, it may be 
proposed that testosterone, by increasing the rate of biotransformation 
to the epoxide, increases the toxicity of Heptachlor, while SKF-525-A, 
through its inhibitory action on this reaction, delays the appearance 
of toxic symptoms. One point which must not be overlooked, however, is 
the probability that, only the rate of metabolism is effected by these 
compounds, and that some metabolic conversion of Heptachlor will occur 



58 



regardless of the endocrine condition of the animal. In addition, there 
is no evidence that Heptachlor per se is inactive, and it is quite 
possible that the toxic effects observed in a particular animal at a 
given time are produced by the sum of the concentrations of both Heptachlor 
and its epoxide present at that time. 

The action of propylthiouracil in speeding the onset of toxic 
reactions and increasing the susceptibility of rats to Heptachlor was 
unexpected, since PTU induced hypothyroidism normally slows metabolism, 
thereby prolonging pentobarbital sleeping time in rats (34), an effect 
shared by SKF-525-A. Since evidence which had been obtained in previous 
studies indicated that a decrease in metabolism resulted in a decreased 
toxicity, a possible explanation for this increase in toxicity could come 
from an action of PTU on some other organ or enzyme system which increases 
the animals susceptibility to Heptachlor or its epoxide. It is also 
possible that a pathway of metabolism of Heptachlor which has not yet 
been described may be effected. There is no experimental evidence, 
however, to support either possibility. 



CHAPTER V 

LOCALIZATION OF THE SITE OF ACTION OF HEPTACHLOR 

Review of the Literature 
There are no reports in the literature concerning pharmacological 
investigations of Heptachlor. Studies with Aldrin (8) and Dieldrin (9) 
have indicated that these compounds produce an apparent potentiation of 
acetylcholine, and an increase in the sensitivity of spinal centers to 
this compound. However, neither a direct peripheral effect nor inhibi- 
tion of cholinesterase could be demonstrated for these two cyclodiene 
insecticides. From these studies, it was concluded that the parasympa- 
thetic activity as well as the convulsant properties of Aldrin and 
Dieldrin are of central nervous system origin. 

Since the principal pharmacologic effects produced by Heptachlor, 
and other members of the cyclodiene family, seem to consist chiefly of 
stimulation of the central and autonomic nervous systems, pharmacologic 
investigation of this compound was confined to those areas. Localization 
of the principal site of activity in the central nervous system and the 
nature of the autonomic stimulation were the objects of this phase of 
the investigation. 



59 



60 



Experimental 
Peripheral Effects 
The Effect of Heptachlor on Blood Pressure and Respiration 
Materials and Methods 

The effect of Heptachlor on blood pressure and respiration was 
tested on rabbits, cats, and dogs in the following manner: 

Blood pressure measurements were taken from a polyethylene 
cannula introduced into the right femoral artery. The system used for 
these measurements consisted of a Sanborn electromanometer model 121-B- 
100, a Sanborn DC amplifier model 64-300B and a Sanborn Twin-Viso 
recorder model 60-1300B. The femoral vein of the opposite leg was 
cannulated to provide a site for intravenous injection of Heptachlor or 
other compounds. All animals were anesthetized with pentobarbital sodium, 
35 mg/Kg intraperitoneally and were given additional pentobarbital 
intravenously as needed. 

The amplitude and frequency of respiration, recorded only for 
dogs, was accomplished by using a displacement transducer, attached by 
means of a silk suture to the skin of the chest, a few centimeters above 
the xyphoid process. A Sanborn strain gauge amplifier, model 64-500B 
and a Twin-Viso recorder comprised the remainder of this system. 

Heptachlor emulsion, 40 mg/ml was given in 1 ml doses initially, 
and in some experiments in increasing amounts up to 10 ml until signs 
of central nervous system stimulation appeared. When tremors or 
convulsions developed, pentobarbital sodium was given intravenously until 
they were controlled. 



See Appendix II for the method used to prepare the emulsion. 



61 



The dogs used in these experiments were healthy 8 to 12 Kg 
mongrels. Healthy alley cats weighing 2 to 4 Kg, and white albino 
rabbits which weighed 2 to 3 Kg were also used in these studies. 

Results 

Heptachlor, given in amount sufficient to produce tremors or 
convulsions, produced no perceptible change in the blood pressure in 
the four rabbits tested. The total amount of Heptachlor necessary to 
produce these symptoms was dependent on the depth of anesthesia of the 
animal being tested and varied from four injections of 40 mg, spaced at 
two-minute intervals, to nine such injections. In trials on three 
unanesthetized rabbits, convulsions, terminating in death, were produced 
by a single injection of 40 mg of Heptachlor. Convulsions in anesth- 
etized animals were easily controlled by cautiously injecting pento- 
barbital sodium intravenously until the convulsions or tremors ceased. 
This was usually accomplished by 1 to 2 ml of a 25 mg/ml solution of 
pentobarbital. 

The initial blood pressure studies on cats showed that a profound 
hypotensive effect was produced by the Heptachlor emulsion; however, the 
control emulsion produced a similar drop in blood pressure. This fall 
in pressure was found to be due to a constituent of the lecithin used 
to prepare the emulsion. This constituent may be removed by a series 
of extraction with organic solvents and the success of this extraction 
procedure may be demonstrated by the failure of the purified product to 



The method of preparation of the control emulsion is found in Appendix 



62 



produce a fall in the blood pressure of a barbitalized cat (46). Due to 
the expense and complicated nature of this procedure, purification of 
the lecithin used in our emulsions was not attempted and blood pressure 
studies in the cat were discontinued. 

Dogs normally did not show a fall in blood pressure after injec- 
tions with emulsions containing lecithin. All dogs used in these 
experiments were given 10 ml of the control emulsion intravenously at 
the beginning of the experiment to determine if they would react to the 
hypotensive constituent of lecithin. Of eight animals tested only one 
showed the drop in blood pressure characteristic of the effect produced 
in cats. The recording of this blood pressure effect is found in 
Figure 10. This animal was not used for blood pressure and respiration 
studies. 

Heptachlor induced no change in the blood pressure of dogs, when 
given in an amount producing symptoms of central nervous system 
stimulation. In some instances, however, a fall in pressure did develop 
when convulsions were allowed to continue for a few minutes. This fall 
was probably secondary to the convulsions rather than a direct effect of 
Heptachlor since a pressure drop did not precede any convulsive episode, 
but was frequently seen if the convulsions were allowed to continue for 
a period of time. The transitory drop seen in Figure 11 could be 
duplicated by injection of a similar volume of the control emulsion. 

The intravenous administration of 400 mg of Heptachlor produced 
a sustained increase in respiratory rate in all animals tested. After 
a short delay, most animals began rapid, shallow breathing, which 
gradually increased in depth while maintaining the increased frequency. 



63 




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A second injection produced an additional increase in respiration which 
followed a similar pattern. In deeply anesthetized animals the first 
injection did not produce a rapid shallow breathing, but groups of two 
to three respirations which occurred at about the same frequency as the 
single respiration before Heptachlor was injected. Subsequent injections 
produced the type of rapid shallow breathing previously described. These 
effects are shown in Figure 11. Respiratory stimulation persisted and 
in most cases the animal developed tremors, f asciculations , and convul- 
sions. The latter effects may easily be controlled with an intravenous 
injection of pentobarbital 25 mg/ml, administered cautiously. However, 
this procedure must be repeated frequently as the stimulating effects of 
Heptachlor may appear again after five to ten minutes. Attempts to lower 
the respiratory rate with pentobarbital in the two cases attempted ended 
in death of the animals, before the original rate of respiration had been 
reached. Attempts to restore respiration in these animals by the immediate 
injection of 400 mg of Heptachlor were unsuccessful. 

These experiments indicate that Heptachlor shares the respiratory 
stimulating properties of other members of the cyclodiene group, but 
lacks the vasodepressive and bradycardia producing effects produced by 
Aldrin and Dieldrin. 

The Action of Heptachlor on Isolated Rabbit Ileum 
Materials and Methods 

To determine if a parasympathetic response to Heptachlor could be 
demonstrated in smooth muscle, sections of ileum, 2 to 3 cm long, taken 
from a site proximal to the cecum were removed from rabbits freshly 







66 



killed by a blow on the head. This area of the intestine is recommended 
by Ludeuna (47, p 142), who states that motility and durability decrease 

from the cecum to the duodenal end of the small intestine. The intestine 

o 

strips were placed in warm Ringer's solution at 35 to 38 C, and 

constantly aerated. Activity was recorded by means of a displacement 
transducer connected to a Sanborn DC amplifier and Twin-Viso recorder, 
as previously described for the recording of respiration. When amplitude 
and frequency of contraction became stabilized, 1 ml of the control 
emulsion was introduced into the bath and allowed to remain in contact 
with the intestinal strip for five minutes. Immediately following this 
period, 1 ml of the Heptachlor emulsion containing 40 mg/ml was added 
to the bath and allowed to remain in contact with the intestinal strip 
for five to thirty minutes. At this time, all of the solution was 
drained from the bath and replaced with fresh, aerated solution. The 
preparation was then tested for its ability to respond to acetylcholine 
by adding enough of the latter compound to the bath, to achieve a final 
concentration of 1 ppm. 

Results 

Amplitude, frequency and tone level of these preparations were 

not altered by contact with Heptachlor for periods of time up to thirty 

minutes. All strips were found to be reactive to acetylcholine following 

exposure to Heptachlor. A typical recording obtained in this procedure 

is found in Figure 12. 













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68 



Isolated Lung Perfusion 
Materials and Methods 

In an effort to determine what part, if any, of the respiratory 
difficulty exhibited by animals acutely poisoned with Heptachlor was due 
to bronchiolar constriction, isolated rabbit lungs were perfused, using 
the method of Sollmann and Von Oettingen (48), described as follows: 
A Mariotte bottle filled with Locke-Ringer's solution was connected by 
means of a rubber tube to a Woulff bottle, also filled with Locke-Ringer's 
solution and maintained at 40 C with a water bath. The outflow of the 
Woulff bottle was attached by means of a short rubber tube to a "T" 
tube. One free end of the "T" tube was connected by means of a short 
piece of rubber tube to a cannula tied into the trachea, and the other 
end to a short section of rubber tubing which was occluded by means of 
a pinch clamp. 

After expelling the air from the lungs, the level of the Mariotte 
bottle was adjusted so that air entered the bottle at a rate of about 
twenty bubbles per minute. When this rate appeared to be stabilized, 
the preparation was considered ready for use. To determine the effect 
of Heptachlor on the perfusion rate of this preparation, 1 ml of Heptachlor 
emulsion containing 40 mg of Heptachlor was injected into the rubber 
tubing, just above the trachea. Immediately following this, 1 ml of 
perfusion fluid was removed from the tube connecting the Mariotte 
bottle to the Woulff bottle in order to maintain a constant perfusion 
pressure. The rate of bubbles entering the Mariotte bottle per minute 
were counted every two minutes, for a total of ten times. At this time 
1 ml of pilocarpine nitrate, 1:1000 solution, was introduced into the 



69 



trachea as previously described, and after five minutes the inflow of 
bubbles again recorded for one minute to determine if the preparation 
would respond to an agent known to produce bronchial constriction. 
Prior to injection of Heptachlor emulsion, an injection of the control 
emulsion was made to determine if the perfusion rate could be changed 
by the emulsion alone. 

Results 

The rate of perfusion was not altered by either the control or 
Heptachlor emulsion in the four preparations tested. Pilocarpine nitrate 
tested on the same lungs produced an average drop in perfusion rate of 
above 50 per cent. Data obtained from these experiments indicate that 
Heptachlor has no direct effect on the bronchial muscles and that 
respiratory difficulties observed in acutely poisoned animals are probably 
not of a peripheral nature. 

Effect on the Frog Rectus Muscle 
Materials and Methods 

The purpose of this experiment was threefold: 1) to determine if 
Heptachlor had a direct effect on skeletal muscle; 2) to determine if 
Heptachlor would influence the response of skeletal muscle to acetyl- 
choline; and 3) to determine if Heptachlor could inhibit cholinesterase 
in vitro . 

The rectus abdominus muscle of the frog was removed and set up 
for recording of contractions according to the method of Burn (49, p 1). 
Contractions were recorded using a displacement transducer and Sanborn 
Twin-Viso recorder as previously described for smooth muscle. For parts 



70 



1 and 2, contraction was obtained by adding 1 ppm acetylcholine to the 
bath in which the muscle was immersed. This drug was allowed to act on 
the muscle for ninety seconds and the height of contraction recorded on 
a stationary drum. At this time the solution was drained from the bath 
and fresh frog Ringer's solution was added. When the muscle had returned 
to its original length, the drum was advanced 0.5 cm and stopped again 
for the next recording. The preparation was allowed to rest five minutes, 
at which time 40 mg of Heptachlor, contained in 1 ml of the emulsion was 
added. This emulsion was allowed to remain in contact with the muscle 
for a period of five minutes and at this time acetylcholine, 1 ppm, was 
added and allowed to act on the muscle for ninety seconds as previously 
described. 

To determine if cholinesterase could be inhibited by Heptachlor 
(Part 3), 0.2 ml of horse serum, containing this enzyme was incubated 
for five minutes with 1 ml of 1:100,000 acetylcholine solution as a 
control. The same procedure was followed after a ten-minute period of 
incubation of the horse serum with 1 ml of Heptachlor, 40 mg/ml. Control 
contractions produced by 1 ml of 1:100,000 acetylcholine solution added 
to the bath were recorded, initially and following the addition of the 
last test solution to the bath, to indicate the responsive state of the 
muscle. The solutions containing the acetylcholine and the serum 
mixtures, were allowed to react with the muscle for ninety seconds and 
the bath was drained and fresh frog Ringer's solution added, immediately 
following each test. The muscle was allowed five minutes to recover 
before the next test was performed. The order in which the tests were 
performed was: 1) acetylcholine, 2) acetylcholine plus horse serum, 



71 



3) horse serum containing Heptachlor plus acetylcholine, and 4) acetyl- 
choline. A new muscle was used for each series. 

Results 

In the four rectus abdominus preparations tested, Heptachlor 
showed neither direct action on the muscle, nor potentiation of the 
response to a standard concentration of acetylcholine. 

Cholinesterase of horse serum was not affected by incubation with 
Heptachlor, and no response was obtained when the horse-serum-Heptachlor- 
acetylcholine mixture was added to the muscle bath. Inactivation of 
acetylcholine was complete in the mixture containing horse serum and 
Heptachlor, and in the mixture containing horse serum alone. 

Central Effects 

Localization of the Site of Action in the Central Nervous System of 
the Frog 

Materials and Methods 

For beginning studies, the frog is an ideal experimental system, 
since discrete areas of its central nervous system may be removed quickly 
and easily with the preparation surviving for several hours after the 
operation. 

Decerebration is performed by severing the head of the frog with 
a pair of scissors at the level of the posterior margin of the eyes. In 
order to remove the optic lobes, the part corresponding to the tectum of 
the midbrain in higher animals, decerebration was first performed as 



Frogs used in these studies were Rana pipiens , obtained from J. R. 
Schettle, Stillwater, Minnesota. 



72 



above, and the optic Lobes, exposed by this procedure, were removed with 
a blunt probe. A third type of preparation produced by removing the 
entire brain was obtained by severing the head with scissors just caudal 
to the posterior border of the tympanic membrane, resulting in a spinal 
animal (50, p 256). 

This experiment was performed in two parts: 1) determination 
of the level of transection of the brain necessary to prevent development 
of convulsion and 2) determination of the level of transection of the 
brain necessary to abolish convulsions, once they had developed. For 
Part 1, four groups of three frogs each were placed in battery jars 
each containing a small amount of frog Ringer's solution. Groups of 
three decerebrate, optic lobectomized, and spinal frogs were prepared 
as previously described, the remaining group of three frogs served as 
unoperated controls. After about fifteen minutes, each frog was given 
25 mg of Heptachlor in corn oil by injection into the ventral lymph sac. 
These frogs were then observed for twelve hours for appearance of 
convulsions. 

The procedure for Part 2 was essentially the same as Part 1 
except that the operations were not performed until convulsions began 
to develop. Frogs were divided into four groups of three frogs each 
and given 25 mg of Heptachlor in corn oil by injection into the ventral 
lymph sac. The frogs were then placed in battery jars which were 
labled as to the type of operation which would be performed in the event 
that convulsions did develop. The frogs were observed for a period of 
twelve hours and the appropriate operation performed when indicated. 



73 



Results 

The development of convulsions in normal frogs required from six 

to ten hours, and consisted of a series of clonic convulsions terminating 
in a tonic convulsion of a few seconds duration. This pattern was 
repeated following periods during which the frog appeared normal. The 
same activity was present in decerebrate and optic lobectomized frogs, 
but not in spinal preparations. 

The abolition of convulsions in frogs was successful only in the 
spinal preparation, but, in general, this procedure was not as successful 
as the previous experiment. Since the appearance of convulsions develop 
over a long period of time, some operations were not performed until the 
symptoms were well advanced, and some of these animals did not recover 
from their surgical treatment. Observations on the successful preparations, 
however, showed that the convulsions could be stopped by sectioning below 
the brain stem, but not by removal of the cerebrum or optic lobes. These 
experiments indicate that the principal site of action of Heptachlor in 
the frog is in the midbrain. 

Localization of the Site of Action in the Central Nervous System of Rats 
Materials and Methods 

In an effort to determine the principal site of convulsive 
activity of Heptachlor in warm-blooded animals, a series of decerebrate- 
spinal rats was prepared, using a modification of the separate methods 
of decerebration and production of spinal animals of D' Amour and Blood 
(44, p 53, 54) as described below. 

One day prior to use in the site of action studies, the rats 
were anesthetized with chloral hydrate, 400 mg/Kg, and the spinal cord 



74 



exposed between the sixth and ninth thoracic vertebra. Tension was 
applied to the tail, and the cord transected with a scapel„ The cord 
was checked for completeness of section and the incision closed with 
silk suture and Michael clips. Spinal section at this level does not 
interfere with respiration. 

The same animal was then prepared for decerebration by making a 
mid-line incision over the region of the cerebellum. A hole was drilled 
just caudal to the transverse sinus, until the dura could be seen. This 
hole was then filled with bone wax and the incision closed with Michael 
clips. 

A third operative procedure performed at the same time consisted 
of disecting the trachea free from overlying muscle and surrounding 
fascia, so that a tracheal cannula could later be inserted in a short 
period of time. 

These procedures resulted in an animal which could be decerebrated, 
and in which a tracheal cannula could be installed in less than two 
minutes. In addition, spinal reflex activity had returned to the areas 
below the section on the day following the operation and the animal was 
suitable for the demonstration of the effects of drugs on spinal activity. 

On the day following the preparatory operations the animal was 
lightly anesthetized with ether and the clips were removed from the 
head, exposing the hole caudal to the transverse sinus. A blunt probe 
was inserted directly downward and moved from side to side several times 
in order to insure complete decerebration. Sectioning at this level is 
said to separate all connection rostral to the pons (44, p 54). The 
animals were turned on their backs, and the trachea rapidly exposed and 
cannulated, using polyethylene tubing of appropriate size. 



75 






The animals were then observed for the appearance of decerebrate 
rigidity which appeared, in successful experiments, in five to fifteen 
minutes. This was comprised chiefly of extensor rigidity of the fore- 
limbs, as the hindlimbs were previously severed from their cerebral 
connections by spinal sectioning. 

When the animal's respiration appeared to be stabilized, a test 
dose of 0.5 ml of the control emulsion, was administered via the 
sublingual vein, and the animal was observed for effects produced by the 
emulsion alone. After two minutes, a dose of 0.5 ml of the Heptachlor 
emulsion containing 20 mg of Heptachlor was given in a similar manner. 

The response to Heptachlor given under these conditions could 
fall into three broad categories: 

1) No effect, indicating origin of the convulsive activity lay 
in the cerebrum. 

2) Convulsive activity in the areas still innervated by the 
brain stem and upper spinal cord, and not in the areas served by the 
spinal cord distal to the section, indicating that principal activity 
lay in the brain stem. 

3) Convulsive activity both above and below the cord section, 
indicating direct action of the spinal cord itself, not dependent on 
higher centers. 

This method has the advantage that the site of action may be 
found using a single animal instead of two, as would be required for 
separate decerebrate-spinal animals. One serious drawback, however, 
is a high number of unsuccessful experiments, since only six out of 
twenty attempted preparations developed signs of decerebrate rigidity 



76 



and were considered acceptable for use in these experiments. In the 
other animals, respiration ceased shortly after decerebration and the 
animals died if they were not maintained on a respirator pump. These 
animals were considered unsatisfactory for use. 

Results 

Immediately following the injection of Heptachlor, a marked 
increase in rate and depth of respiration appeared. After three to five 
minutes, the convulsant effects of this compound could be seen through 
the decerebrate rigidity already present in the animal. These effects 
consisted of tremors, fasciculations and clonic spasms of the muscles 
of the upper extremities. Occasionally these spasms would terminate in 
a prolonged tonic extension. During intervening periods, the animal 
returned to a state of decerebrate rigidity. 

There was no increase in tone or spontaneous activity in the 
muscles of the hindlimb of the animal indicating an absence of spinal 
or direct skeletal muscle effect of Heptachlor; 

All animals were dead within one hour after injection of Heptach- 
lor. Respiration ceased before cardiac arrest occurred. 

From these experiments it can be concluded that in the rat, as 
in the frog, the principal site of activity of Heptachlor lies in the 
brain stem. It is probable, however, that Heptachlor, as is the case 
of other centrally acting compounds, effects other areas of the central 
nervous system, and its classification as a brain stem stimulant is 
more one of convenience than precise anatomical localization. 






77 



Discussion 

The two pharmacologic properties described for Heptachlor — 
central nervous system stimulation and parasympathomimetic activity -- 
apparently have similar origins. The absence of a response in vivo by 
tissues which respond readily to autonomic drugs leads to the conclusion 
that direct stimulation of autonomically innervated structures plays no 
part in the symptoms of acute Heptachlor poisoning. 

No evidence favoring the inhibition of cholinesterase, direct 
action on skeletal muscle, or potentiation of acetylcholine by Heptachlor 
was obtained on the isolated frog rectus muscle. These findings, coupled 
with the observation that increased secretory activity in rats was most 
prominent following periods of convulsions indicates that the central 
nervous system is the origin of both convulsive and autonomic effects 
of Heptachlor. This conclusion was also reached in studies of Dieldrin 
(9) and Aldrin (8). However, these compounds were reported to have 
produced bradycardia, vasodepression and potentiation of acetylcholine. 
None of these effects were seen with Heptachlor. 

Respiratory stimulation was the most consistent effect produced 
by Heptachlor in normal-anesthetized and decerebrate animals. This 
effect became apparent immediately following the intravenous injection 
of large doses of this compound. The cumulative nature of this compound 
is demonstrated by the appearance of symptoms after a series of 
injections which, individually, would not provoke a response. Respir- 
atory stimulation could be increased further by additional injections 
of Heptachlor. Tremors and convulsions which usually followed the 
initial respiratory stimulation were easily controlled by intravenous 



78 



pentobarbital. However, respiratory rate was more difficult to control. 
The deaths of two animals which resulted during attempts to restore the 
respiratory rate to normal suggest that efforts to antidote Heptachlor 
by intravenous barbiturates must be made with extreme caution. This 
sudden cessation of respiration in animals which had been hyperventilat- 
ing as a result of the stimulating effects of Heptachlor, may be due to 
a sudden removal of this stimulation by the barbiturates, leaving a state 
of fatigue brought on by the prolonged over-activity. Additional 
contributory factors would be a lack of the normal carbon dioxide 
stimulating effect, since hyperventilation would be expected to result 
in a low partial pressure of circulating carbon dioxide. The blood of 
such an animal would also be well oxygenated, and an additional means of 
respiratory stimulation would not be available. Respiration arrested in 
the manner described was not restored by additional Heptachlor injections. 

The principal locus of activity of Heptachlor, in the frog and 
rat, is the brain stem. In the frog, removal of the cerebrum failed to 
stop or prevent the convulsions following injections of Heptachlor, 
while removal of the entire brain successfully blocked the appearance 
of convulsions. In these respects, and in the general appearance of 
the convulsion, Heptachlor is similar to the brain stem stimulants 
such as Metrazole. However, no central nervous stimulant has its effects 
confined to a discrete area of the central nervous system, but different 
areas of the brain seem to vary in susceptibility to a given dose. 
Thus Metrazole, a typical brain stem stimulant, in high doses will also 
stimulate the spinal cord. In view of the many interconnecting pathways 
of the central nervous system it is impossible to assign discrete areas 



79 




of the central nervous system as the site of action of Heptachlor, or any 
other central nervous system stimulant. The respiratory center of the 
medulla seems to be especially sensitive to Heptachlor as the effects of 
stimulation are first to appear in this area and are not controlled with 
measures used successfully for the control of convulsions and tremors. 

Evidence favoring medullary action rather than action of the 
carotid sinus as the cause of respiratory effects of Heptachlor, arises 
from the absence of inspiratory gasps characterized by compounds such 
as lobeline or cyanide which stimulate this area directly (23, p 298). 






CHAPTER VI 



SUMMARY AND CONCLUSIONS 






The LD50 values for male and female rats determined in this 
study were 59 and 132 mg/Kg respectively. No rats from either sex 
survived the first two weeks of a six-month chronic feeding period, 
when fed a diet containing 1 or 0.1 per cent Heptachlor. During the 
remainder of this period, female rats proved to be more susceptible to 
the lethal effects of the higher concentration of Heptachlor than male 
rats. 

Significant weight changes were found in the liver of male and 
female rats fed 0.01 per cent and 0.001 per cent Heptachlor. A signifi- 
cant change in kidney weight was also noted with the higher concentration 
in both sexes. An increase in testicle weight was found in male rats 
fed 0.01 per cent Heptachlor. 

Pathological findings of tissues from the organs of Heptachlor 
fed animals consisted chiefly of degenerative changes of the liver. 
These changes were much more extensive in female than in male rats. 

An endocrine basis for the difference in sex response was found 
using castrate and hormone treated animals. Castration delays the 
appearance of toxic symptoms of Heptachlor in male rats, while testoste- 
rone treated rats developed symptoms more rapidly than other groups. 
Estradiol was without apparent effect on the toxicity of Heptachlor in 
either sex. 



80 



81 



Evidence suggesting metabolic activation of Heptachlor was 
obtained from experiments showing that the appearance of toxic symptoms 
of Heptachlor could be accelerated by testosterone pretreatment and 
delayed by SKF-525-A, compounds known to speed up and retard respectively, 
the rate of biotransformation of a number of compounds. 

Propylthiouracil, given in the diet of male and female rats 
increases the speed of onset and mortality of Heptachlor in the female 
and to a lesser extent, the male. 

Heptachlor was shown to have no direct effect on isolated smooth 
or skeletal muscle. No potentiation of acetylcholine or inhibition of 
cholinesterase could be demonstrated, and blood pressure was not effected. 

Heptachlor given intravenously produced a sustained increase in 
the respiratory rate in both decerebrate and normal anesthetized animals. 
This stimulation appeared before other symptoms of central nervous 
system stimulation could be seen. 

Muscle tremors and convulsions of Heptachlor poisoned animals are 
easily controlled with pentobarbital sodium, but frequent administration 
are required, due to the persistance of Heptachlor stimulation. Respira- 
tion stopped with pentobarbital in Heptachlor poisoned animals could not 
be reinitiated by this compound. 

The activity of Heptachlor in frogs and rats was found to be 
confined to the central nervous system, with the principal site of action 
being the brain stem. This finding coupled with failure of Heptachlor 
to produce any peripheral response indicates that both the convulsive 
and autonomic effects of Heptachlor are mediated through the central 
effects of this compound. 



> 



» 



APPENDICES 



> 



APPENDIX I 

PREPARATION OF THE CORN OIL SOLUTION OF HEPTACHLOR 

Usual organic solvents in which Heptachlor is soluble are either 
toxic or have undesirable pharmacologic activity. Since Heptachlor is 
insoluble in water, it was necessary to use a vegetable oil as the 
solvent for oral administration in the acute toxicity studies. Corn 
oil was selected because of its ready availability. The solutions were 
prepared by dissolving a carefully weighed amount of Heptachlor in a 
small amount of hot corn oil and then diluting to volume. In all cases 
the quantity of drug to be given per kilogram of body weight was 
present in 5 ml of solution. 



83 



APPENDIX II 



PREPARATION OF THE HEPTACHLOR EMULSION FOR 
PARENTERAL AND ISOLATED TISSUE STUDIES 



For intravenous administration and isolated tissue studies, the 

oil solution of Heptachlor was unsuitable because of its immisciblity 

with physiologic media. Organic solvents, for reasons previously 

stated, were also unsuitable. To solve this problem, an emulsion 

similar to one used for intravenous administration of lipids to humans 

(46) was prepared. The formula of this emulsion is listed below: 

Corn oil containing 207» w/v Heptachlor 20 ml 

Lecithin 1 Gm 

Pluronic Ffcs 0.2 Gm 

Normal saline 80 ml 

To prepare the emulsion, the lecithin was dissolved in the corn oil 
solution of Heptachlor, and this solution was slowly stirred into the 
saline in which the Pluronic Fgg had previously been dissolved, The 
resulting crude emulsion was then passed five times through a Manton- 
Gaulin two-stage laboratory homogenizer at a pressure of 2500 pounds 
per square inch. This procedure is stated to result in a very stable 
emulsion, miscible with physiologic media, having a particle size of 
one micron or less (46) and containing 40 mg/ml of Heptachlor. A 
control emulsion containing 20 per cent pure corn oil was prepared in 
a similar manner. 



84 



APPENDIX III 

CALCULATION OF THE LD 50 BY THE LITCHFIELD 
AND WILCOXON METHOD 

Calculation of the LD^q by this method is accomplished by first 
plotting all responses exclusive of the and 100 per cent effects on 
log probability paper (Codex 3128). A line is then fitted to these 
points by inspection. The corrected and 100 per cent effects are 

obtained using information obtained from the graph and a nomograph 

o 
contained in the original article. A (Chi) test is then performed to 

determine if the line fits the data. This method also provides a means 

for the determination of the 95 per cent confidence limits of the LD 50 

and the slope of the line. The original paper presenting this method 

of calculation contains several nomographs and tables which greatly 

simplify the calculations (21). 

Male Rats 

Total contribution of (Chi) 2 from Table 5 - 0.335 

Total number of animals - 48 

Number of doses ■ K ■ 6 

Animals/dose ■ 48/6 - 8 

(Chi) 2 - 0.335 x 8 - 2.680 

Degrees of Freedom - N - K-2 - 4 
(Chi) 2 for N - 4 is 9.49. Since 2.680 is less than 9.49, the data are 
not significantly heterogenous and the line is a good fit. 



85 






86 



From the graph of LD.q the following values were found: 

LD 84 - 84 mg/Kg 

LD 5Q - 59 mg/Kg 

LD,, - 42 mg/Kg 

Calculation of the Slope Function S 
_ LDo./LD^ + LD^/LD 



84' 50 ""50' ""16 « 1.415 
2 

Calculation of the confidence limits of the LD 5Q for 19/20 probability 

limits 

LDc n x F T _ ■ upper limit 
:>U LU 5Q 

^50 / F LD 50 " lower limit 

r u> R -s 2 - 77 /VF 

50 
N* ■ total number of animals tested at those doses whose expected 

effects were between 16 and 84 per cent » 24 

Fld - 1.415 2 ' 777 VK- 1.2 






50 
LD 5Q x Fjjj - 59.0 x 1.2 - 70.8 mg/Kg 

LD 50 / F^ - 59.0 / 1.2-49.1 mg/Kg 



Summary 



LD 50 = 59(70.8 to 49.1) mg/Kg 
S = 1.415(1.14 to 1.175) 



Female Rats 

The data obtained from the determination of the LDcq are given 
in Table 6. Calculations were carried out in the manner previously 
described, and only the results are given. The graph for these data 
is found in Figure 8. 



87 



Total number of animals ■ 48 

Number of doses ■ K • 6 

Animals/dose ■ 8 

Total contribution to (Chi) 2 from Table 6 - 0.625 

(Chi) 2 for 6 degrees of freedom «= 12.6. Since 5.0 is less than 12.6, 

the line is a good fit. 

The following values were obtained from the graph: 

LD - 195 mg/Kg 
84 

LD - 132 mg/Kg 

LD,, - 95 mg/Kg 
The calculated 19/20 probability limits for the slope function were: 

upper limit ■ 1.85 

lower limit - 1.17 
The calculated 19/20 probability limits for the LD 5Q were: 

upper limit ■ 154 

lower limit "114 
The value of the slope function was found to be 1.475 
Summary of data: 

LD 5Q - 132(114 to 154) mg/Kg 

S - 1.475(1.85 to 1.17) 



BIBLIOGRAPHY 



1. Smyth, Henry P., Jr., Agric. Food Chem. , 4:644 (1956). 

2. Frear, Donald E. H. , "Chemistry of the Pesticides," 3rd ed. , D. Van 

Nostrand Co., New York, N. Y., 1955. 

3. Metcalf, R„ L. , "Organic Insecticides," Interscience Publishers, Inc., 

New York, N. Y. , 1955. 

4. Derbes, V. J., et al . , J. Am. M. Assoc , 158:1367 (1955). 

5. Negherbon, W. 0. (ed.), "Handbook of Toxicology," Vol. Ill, W. B. 

Saunders Co., Philadelphia, Pennsylvania, 1959. 

6. McGee, L. C. , and Reed, H. L. , J. Am. M. Assoc , 149:1124 (1952). 

7. Leman, A. J., Quart. Bull. Assoc Food Drug Of fie. U. S. , ^5:122 

(1951). 

8. Gowdey, C, et al., Can. J. Med. Sci. , 30:520 (1952). 

9. Gowdey, C, et. al . , Can. J. Biochem. Physiol. , 32:498 (1954). 

10. Brown, A. W. A., "Insect Control by Chemicals," John Wiley and Sons, 

New York, N. Y., 1951. 

11. Treon, J. F., and Cleveland, F. P., J. Agric. Food Chem. , 3:842 (1955) 

12. Radeleff, R. D., Vet. Med. (Chicago, Illinois), 46:305 (1951). 

13. Lehman, A. J., Quart. Bull. Assoc. Food and Drug Of fie l), S. , 16:3 

(1952). 

14. "Summation of the Toxicity of Heptachlor to Warm-Blooded Animals," 

(Mimeographed), The Velsicol Chemical Corp., Chicago, Illinois. 

15. Barnes, J. M. , and Denz, F. A., Pharmacol. Rev. , 6:191 (1954). 

16. Johnston, E. , et al., J. Animal Sci. , 2:244 (1943). 

17. Malewitz, T. D., and Smith, E. M. , Stain Tech. , 30:311 (1955). 

18. "Manual of Histologic and Special Staining Techniques," 2nd ed., 

The Blakiston Division, McGraw-Hill Book Co., New York, N. Y. , 
1949. 



88 



89 



19. Bernstein, L. , and Weatherall, M. , "Statistics for Medical and Other 

Biological Students," E. and S. Livingstone, Ltd., Edinburg, 
Scotland, 1952. 

20. Anderson, W. A. D., "Synopsis of Pathology," The C. V. Mosby Co., 

St. Louis, Missouri, 1957. 

21. Litchfield, J. T. , Jr., and Wilcoxon, F., J. Pharmacol. Exptl. 

Therap. , 96:99 (1949). 

22. Seyle, H. , J. Pharmacol. , 95:79 (1949). 

23. Drill, V. A. (ed.), "Pharmacology in Medicine," 2nd ed., McGraw- 

Hill Book Co., Inc., New York, N. Y., 1958. 

24. Davidow, Bernard and Radomski, J. L. , J. Pharmacol. Exptl. Therap. , 

103:259 (1953). 

25. Radomski, Jack L. , and Davidow, Bernard, ibid. , 107_:266 (1953). 

26. Davidow, Bernard, Fed. Proc. , 10:291 (1951). 

27. Perry, A. S., et_al. , J. Econ. Ent. , 53:346 (1958). 

28. Jarcho, L. W. , et al. , Proc. Soc . Exptl. Biol. Med. , 74:332 (1950). 

29. Hoick, H. G. 0. , et al. , J. Am. Pharm. A. Sci. Ed. , 31;116 (1942). 

30. Cameron, G. R. , et_ al . , J. Path. Bacteriol. , 60:239 (1948). 

31. Tureman, J. R. , et al., Fed. Proc , 13:412 (1954). 

32. Quinn, G. P., et al., ibid ., 13:395 (1954). 

33. Davson, A. N. , Biochem. J. , 61:203 (1955). 

34. Hoick, H. G. 0. , et al. , J. Pharmacol. Exptl. Therap. , 43:276 (1954). 

35. Axelrod, J., et al., ibid. , 112:49 (1954). 

36. Cook, L. , et_ al. , ibid. , 111:131 (1954). 

37. Cook, L. , et al. , ibid. , 112:382 (1954). 

38. Cooper, J. R., et al., ibid. , 112:55 (1954). 

39. La Du, B. N. , and Glaudette, L. , J. Biol. Chem. , 2_14:741 (1955). 

40. Cooper, J. R. , and Brodie, B. B., J. Pharmacol. Exptl. Therap. , 

112:55 (1954). 



90 



41. Fouts, J. R., and Brodie, B. B. , ibid. , 115:68 (1955). 

42. Fouts, J. R., and Brodie, B. B. , ibid. , 116:480 (1956). 

43. Leighty, John A., Personal Communication, March 22, 1961. 

44. D'Amour, Fred E. , and Blood, Frank R., "Manual for Laboratory Work 

in Mammalian Physiology," Rev. ed. , The University of Chicago 
Press, Chicago, Illinois, 1959. 

45. White, A., et al. , "Principles of Biochemistry," 2nd ed. , The 

Blakiston Division, McGraw-Hill Book Co., New York, N. Y., 1959. 

46. Meyer, C. E. , et al. , Metab. , 6:592 (1957). 

47. Bruner, H. D., (ed.), "Methods in Medical Research," Vol. 8, The 

Year Book Publishers, Inc., Chicago, Illinois, 1960. 

48. Sollmann, T. , and Von Oettingen, W. F., Proc . Soc . Exper . Biol. Med. , 

25:692 (1927). 

49. Burn, J. H. , "Practical Pharmacology," Blackwell Scientific Publications, 

Oxford, England, 1952. 

50. Jackson, D. E. , "Experimental Pharmacology and Materia Medica," 

2nd ed., The C. V. Mosby Co., St. Louis, 1939. 




BIOGRAPHICAL SKETCH 

The author was born on May 1, L933 at Holly Springs, Georgia. 
He graduated from Canton High School, Canton, Georgia, in 1950 and 
entered Mercer University the fall of that year. 

He received a B.S. degree in pharmacy from Southern College of 
Pharmacy, Atlanta, Georgia, in 1954 Following graduation he served in 
the army until August, 1956. 

The author began his graduate studies at the University of 
Florida during the Fall Semester of 1956, and has been in attendance 
at that institution since that time. He expects to receive the Ph.D. 
degree in February, 1962. 

The author is a member of Kappa Psi pharmaceutical fraternity, 
Rho Chi honorary pharmaceutical fraternity, Gamma Sigma Epsilon honorary 
chemical fraternity, Phi Sigma honorary biological fraternity and is 
an associate member of the American Association for the Advancement of 
Science. He has been the recipient of a Graduate Council Fellowship 
and is a fellow of the American Foundation for Pharmaceutical Education. 



91 



This dissertation was prepared under the direction of the Chairman 
of the candidate's supervisory committee and has been approved by all 
members of that committee. It was submitted to the Dean of the College 
of Pharmacy and to the Graduate Council and was approved as partial 
fulfillment of the requirements for the degree of Doctor of Philosophy. 



February 3, 1962 



Dean, College of Pharmacy 



Dean, Graduate School 



SUPERVISORY COMMITTEE 



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Chairman 



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UNIVERSITY OF FLORIDA 



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